Tuning microbial populations with programmable nucleases
Various aspects and embodiments of the invention are directed to methods and compositions for reversing antibiotic resistance or virulence in and/or destroying pathogenic microbial cells such as, for example, pathogenic bacterial cells. The methods include exposing microbial cells to a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease targeted to one or multiple genes of interest.
1. A method of detecting a nucleic acid of interest in a viable bacterial cell, comprising delivering to a human subject at least one delivery vehicle that comprises:
(a) a nucleic acid comprising a promoter operably linked to nucleotide sequences encoding a programmable nuclease circuit that induces a double-strand break in the nucleic acid of interest in the bacterial cell in the human subject;
(b) a nucleic acid comprising a RecA promoter or a LexO promoter operably linked to a gene encoding a reporter molecule, wherein the RecA promoter or the LexO promoter activates expression of the reporter molecule in the presence of the double-strand break; and
(c) detecting presence or absence of the nucleic acid of interest based on expression of the reporter molecule in the viable bacterial cell.
2. The method of claim 1 , wherein step (c) comprises detecting transcription or translation of the reporter molecule.
3. The method of claim 1 , wherein the programmable nuclease circuit comprises an RNA-guided nuclease.
4. The method of claim 3 , wherein the programmable nuclease circuit comprises a Cas9 nuclease, a guide RNA (gRNA), and/or a transactivating small RNA (tracrRNA) derived from the Type II CRISPR-Cas system of Streptococcus pyogenes.
5. The method of claim 4 , wherein the gRNA and the tracrRNA form a chimeric single-guide RNA molecule.
6. The method of claim 1 , wherein the programmable nuclease circuit comprises a transcriptional activator like (TAL)-effector nuclease (TALEN) or a zinc finger nuclease (ZFN).
7. The method of claim 1 , wherein the delivery vehicle is a bacteriophage.
8. The method of claim 1 , wherein the delivery vehicle is a phagemid.
9. The method of claim 1 , wherein the delivery vehicle is a donor bacterial cell.
10. The method of claim 9 , wherein the donor bacterial cell is a commensal bacterial cell.
11. The method of claim 10 , wherein the commensal bacterial cell is loaded with at least one conjugative plasmid containing the nucleic acid of (a) and/or (b).
12. The method of claim 11 , wherein the at least one conjugative plasmid comprises a broad-host-range origin of replication.
13. The method of claim 1 , wherein the bacterial cells are pathogenic bacterial cells.
14. The method of claim 13 , wherein the pathogenic bacterial cells are selected from extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli , ESBL Klebsiella pneumonia, Pseudomonas aeruginosa , vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant (MDR) Acinetobacter baumannii , and MDR Enterobacter spp.
15. The method of claim 1 , wherein the nucleic acid of interest includes a mutation.
16. The method of claim 15 , wherein the mutation is a single nucleotide polymorphism (SNP).
17. The method of claim 1 , wherein the nucleic acid of interest comprises a virulence factor gene, a toxin gene, an antibiotic resistance gene, a remodeling gene or a modulatory gene.
18. The method of claim 17 , wherein the nucleic acid of interest comprises an antibiotic resistance gene that confers aminoglycoside resistance, beta-lactam resistance, daptomycin resistance, macrolide-lincosamide-streptogramin B resistance, quinolone resistance, trimethoprim/sulfonamide resistance or vancomycin resistance.
19. The method of claim 1 , wherein the promoter of (a) is an inducible promoter.
20. The method of claim 1 , wherein the reporter molecule is a fluorescent protein.